CN110710450B - Method for improving flavone content in tea through tea leaf botrytis cinerea infection - Google Patents

Abstract

The invention relates to a method for improving flavone content in tea leaves through cladosporium cucumerinum, which comprises the following steps: 1) culturing the alternaria alternata in a culture medium to obtain a spore suspension; 2) and (3) placing the tea leaves in the spore suspension for culturing to obtain the tea leaves infected by the tea leaf botryococcus. The tea leaf spot is a commonly occurring disease in a high-altitude tea area, is difficult to prevent and control, and causes bitter and astringent taste of tea leaves. The application improves the flavonoid content of the tea by infecting the tea leaves with the Cladosporium virens, not only solves the problem of prevention, control and control of high mountain diseases, changes the harm of the tea leaves infected with the Cladosporium virens into the benefit, but also is beneficial to the production of high-flavonoid tea.

Description

Method for improving flavone content in tea through tea leaf botrytis cinerea infection

Technical Field

The invention relates to the field of tea preparation, and in particular relates to a method for improving the content of flavonoid in tea through the infection of tea leaf botrytis cinerea.

Background

Flavonoids generally refer to a series of compounds in which two benzene rings are connected to each other through three carbon atoms. Flavonoids are present in almost all green plants. It is mainly distributed in higher plants, has wide bioactivity, is a strong antioxidant, can effectively remove oxygen free radicals in vivo, prevent cell degeneration and aging, and prevent cancer. The flavone has effects of improving blood circulation, reducing cholesterol, and improving symptoms of cardiovascular disease and cerebrovascular disease. It has good effect on retinopathy and capillary embrittlement caused by diabetes. Can inhibit the exudation of inflammatory biological enzymes, promote wound healing and relieve pain. The tea contains abundant flavonoid glycoside substances, mainly comprises flavonol glycoside, mainly comprises quercetin glycoside, myricetin glycoside and kaempferol glycoside; a small part exists in the form of flavonol aglycone, namely quercetin, myricetin and kaempferol. At present, researches for improving the flavone content are rarely reported in the field of tea preparation.

Disclosure of Invention

Based on the situation, the invention aims to provide a method for improving the content of the flavone in the tea leaves through the infection of the Cladosporium virens.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for improving the flavone content in tea leaves by using Cladosporium virens comprises the following steps:

1) culturing the alternaria alternata in a culture medium to obtain a spore suspension;

2) and (3) placing the tea leaves in the spore suspension for culturing to obtain the tea leaves infected by the tea leaf botryococcus.

In some embodiments, the tea brettanomyces custersii is deposited at the China center for type culture Collection at 7/15/2019 with a deposition number of CCTCC NO: M2019555, and is identified as Elsinoe camelliaa sinesis Cs52, and is deposited at Wuhan university school of eight paths 299 in Wuchang district of Wuhan, Hubei province (the first attached small opposite of Wuhan university), the Wuhan university Collection.

In some embodiments, the culture medium is selected from the group consisting of: PSA medium, Czapek's medium, PDA medium, Bengal red medium and tea juice medium.

In some embodiments, the medium is PSA medium.

In some embodiments, the PSA medium has a pH of 3 to 10.

In some embodiments, the PSA medium has a pH of 7.

In some embodiments, the medium contains a nitrogen source comprising urea, peptone, ammonium nitrate, and glycine, wherein glycine is preferred.

In some embodiments, the medium contains a carbon source comprising maltose, lactose, soluble starch and sucrose, wherein soluble starch is preferred.

In some embodiments, in step 1). The temperature for culturing the tea leaf spot pathogen in the culture medium is 12-34 ℃.

In some embodiments, in step 1). The temperature for culturing the tea leaf spot fungus in the culture medium is 26 ℃.

The tea leaf spot is a commonly occurring disease in a high-altitude tea area, is difficult to prevent and control, and causes bitter and astringent taste of tea leaves. According to the application, the flavonoid content of the tea leaves is improved by infecting the tea leaves with the tea leaf spot, so that the problem of prevention and control of high mountain diseases is solved, the harm of the tea leaves infected with the tea leaf spot is changed into benefit, and the production of high-flavonoid tea leaves is facilitated.

Drawings

FIG. 1 is a picture of visualization of disease of tea inoculated with Cladosporium virens;

FIG. 2 shows the spore and hypha morphology of Cladosporium theophyllum;

FIG. 3 is a micrograph of a section of tea infected with the virus Asterina graminearum;

FIG. 4 is a scanning electron microscope image of tea leaves infected by Asterina graminicola.

Detailed Description

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but rather should be construed as broadly as the present invention is capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Example 1

1.1 Experimental materials

Pathogenic bacteria: the causative bacterium of thea albicans (elsinoe. leucospila).

Tea leaf: is collected from young healthy tender leaves in Ma Pong and diseased leaves infected by tea leaf spot in Shimen Baiyunshan tea field.

1.2 Experimental procedures

1) Collecting the diseased leaves, cleaning with sterile water, cutting diseased tissues from the boundary of diseased leaves to obtain small blocks of about 2mm multiplied by 2mm, soaking in 75% ethanol for 5s, transferring into 3% NaClO solution for sterilization for 110s, transferring into sterile water for cleaning for 3 times, transferring onto sterile filter paper for airing, uniformly placing 5 samples in a culture medium with the diameter of 9cm, and placing in an incubator for dark culture at 24 ℃ for 6d to obtain spore suspension;

2) soaking the collected tea leaves in the spore suspension for 5h, taking out, and culturing in a light incubator (constant temperature of 26 deg.C, humidity of 95%, and light-dark alternation for 12/12 hr) to obtain tea leaves infected with Alternaria virens (see fig. 1-4).

1.3 investigating the growth influence of different factors on the Cladosporium Chachiensis

1) Growth influence of different culture media on Phyllostachys Pubescens

Preparing 5 fungus culture media with different nutrient components including PSA culture medium, Czapek's culture medium, PDA culture medium, Bengal and tea juice culture medium, respectively preparing 4 substituted cane sugars including maltose, lactose, soluble starch and fructose as carbon source culture medium by using Czapek's culture medium as basic culture medium, respectively preparing urea, peptone and NH₄NO₃4 kinds of glycine substituted nitrogen source NaNO₃The medium of (3) and the carbon-deficient and nitrogen-source medium are used as controls. The 6mm cake was punched out with a punch, inoculated in the center of a 9cm diameter plate, cultured continuously at 22 ℃ for 14d, 21d, 28d, and the colony diameter was measured by the cross method, and each treatment set was repeated 4 times.

Analysis of results

As shown in table 1, the results of e.leucospila bacteria grown on different media for 28 days showed that all 5 different media were able to grow but at different growth rates. According to the growth rate from large to small, the growth rate is respectively PSA>Tea juice>Czapek>PDA>Bengal red, with a growth diameter of up to 2.79cm on PSA medium. And then, taking the Czapek's culture medium as a basic culture medium, measuring the colony growth condition of the E.leucospila under the condition of different carbon sources and nitrogen sources, wherein the result shows that the E.leucospila can grow in the culture medium lacking carbon and nitrogen, but the hyphae are fine, the quantity of the hyphae is small, and NH is used for reducing the quantity of the hyphae₄NO₃The utilization rate of the inorganic nitrogen source is the lowest, and the utilization rate of the glycine as the nitrogen source is the highest. The growth results of carbon sources show that, besides lactose and sucrose, other carbon sources are source of E.leucosThe growth of pila is affected, with soluble starch having the greatest effect on the growth of e.

TABLE 1 Effect of different media and carbon and nitrogen sources on E.leucospila growth

2) Growth influence of different temperature and pH on pathogenic bacteria

Temperature influences on the growth rate of pathogenic bacteria: 6mm of the pellet was inoculated into the center of a PSA plate, and cultured at 12 deg.C, 16 deg.C, 18 deg.C, 22 deg.C, 26 deg.C, 30 deg.C, 34 deg.C, 40 deg.C, each treatment was set up for 4 replicates, and the culture conditions and measurement methods were the same as above.

② influence of pH on growth rate of pathogenic bacteria: PSA medium was adjusted to pH 3.0, pH 4.0, pH 5.0, pH 6.0, pH 7.0, pH 8.0, pH 9.0, pH 10.0 with 1mol/L HC1 and NaOH solution, 6mm clumps were inoculated in the center of the plate, cultured at 26 ℃ with 4 replicates of each treatment setup, and the culture conditions and measurement methods were the same as above.

Analysis of results

As shown in Table 2, the results of the growth of the pathogenic bacterium E.leucospila on the culture medium at different temperatures and pH values for 28 days show that the pathogenic bacterium E.leucospila can grow at 12-34 ℃ but the growth rate is not consistent. Wherein the growth rate at 26 ℃ is significantly higher than other temperature conditions, and the growth rate is about 0.11 cm/d. The pathogenic bacterium e.leucospila hardly grows at pH 3, and at pH 7 the growth rate is significantly higher than at other pH values, the growth rate being about 0.10 cm/d.

Table 2 effect of different temperatures, PH on e.leucospila growth

Note: the data in the graph are mean ± s.e. the difference between the different tables for lower case letters after the same column of data is significant (P < 0.05).

1.4 measurement of flavone content

Samples from one bud and one leaf to four leaves are picked in 3 times in 5 months from 2014 to 2016 in the Foding white planting area of Shimen Baiyunshan forest farm, and the samples are classified into CK (incidence rate 0), I (incidence rate 11-40%), II (incidence rate 41-50%), III (incidence rate 51-80%) and IV (incidence rate 81-100%).

Baking the green tea sample: steaming for 2min, drying at 95 deg.C to dry, and storing at 4 deg.C.

According to the principle that the flavonoid compounds in the tea leaves react with the aluminum trichloride to generate yellow complex, and the color depth is in proportional relation with the content of the flavonoid, the total amount of the flavonoid is measured by using an aluminum trichloride colorimetric method. Sucking 0.5ml of tea soup into a volumetric flask, adding 1% AlCl₃10ml of the aqueous solution is shaken up and after 10min, the mixture is subjected to colorimetry by a spectrophotometer in a 10mm cuvette at a wavelength of 420nm and 1% AlCl₃The aqueous solution is blank, and the absorbance E is measured₁. The method has small detection wavelength, and the absorption of tea soup is eliminated by sucking 0.5ml of tea soup into a volumetric flask, adding 10ml of distilled water, shaking, and measuring absorbance E under the same condition after 10min₂. (the content was calculated based on the absorbance equal to 1.00, corresponding to 320ug of flavone)

Total flavone content (%) ═ E₁-E₂)×320×V₁/V₂)/G×100％

In the formula: e₁-sample absorbance; e₂-background absorbance; v₁-volume of extract (ml); v₂-sample volume (ml) at detection; g-sample dry weight (ug).

The results of this study on the total flavonoids in tea samples are shown in table 3. With the increase of disease index, the content of total flavone gradually increases.

TABLE 3 basic ingredient content and significance of differences test results

Note: different lower case letters after the same row data indicate that the difference of different disease incidence levels reaches the significance level (P < 0.05); the minimum detected amount is 0.01mg g.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (9)

1. A method for improving the content of flavone in tea leaves by using Cladosporium virens is characterized by comprising the following steps:

1) culturing the alternaria alternata in a culture medium to obtain a spore suspension;

2) culturing the tea leaves in the spore suspension to obtain tea leaves infected by the Cladosporium virens;

the tea leaf spot fungus is preserved in the China center for type culture collection in 2019, 7 and 25 months, and the preservation number is CCTCC NO: M2019555.

2. The method of claim 1, wherein the culture medium is selected from the group consisting of: PSA medium, Czapek's medium, PDA medium, Bengal red medium and tea juice medium.

3. The method of claim 1, wherein the culture medium is a PSA medium.

4. The method according to claim 3, wherein the PSA medium has a pH of 3 to 10.

5. The method according to claim 3, wherein the PSA medium has a pH of 7.

6. The method of claim 1, wherein the medium comprises a nitrogen source comprising urea, peptone, ammonium nitrate, and glycine.

7. The method of claim 1, wherein the culture medium contains a carbon source comprising maltose, lactose, soluble starch and sucrose.

8. The method of claim 1, wherein in step 1), the temperature for culturing the alternaria tenuissima in the culture medium is 12-34 ℃.

9. The method according to claim 1, wherein the temperature for culturing the cladosporium cucumerinum in the culture medium in the step 1) is 26 ℃.

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